Oxidant stress is implicated in many disorders including ischemia- reperfusion damage, atherosclerosis, pulmonary oxygen toxicity, adult respiratory distress syndrome, and bronchopulmonary dysplasia. Although superoxide dismutases may palliate some of these, the dismutation product of superoxide, hydrogen peroxide, as well as secondarily derived lipid peroxides, also can cause substantial tissue injury. These products are detoxified by the glutathione redox cycle. In models of tolerance to oxidants, profound increases in activities of glucose--phosphate dehydrogenase (G6PD) occur. G6PD is the rate-limiting step of the hexose monophosphate shunt (HMPS) which provides reducing equivalents (NADPH), the fuel for the glutathione redox cycle. The possibility that selective supraphysiologic increases in G6PD can decrease oxidant damage is unexplored. Our hypothesis is that selective increases in G6PD will limit oxidative damage due to peroxides or hyperoxia. The preliminary data supports this hypothesis. Cells transfected with, and transgenic mice bearing, the human G6PD gene will be studied. The effect of G6PD augmentation in cells challenged with elevated fluxes of superoxide, hydrogen peroxide, organic peroxides, or hyperoxia will be determined. Cell damage will be assessed morphologically and biochemically. Hexose monophosphate shunt activity, reduced/oxidized glutathione, NAPDPH/NADP+ ratios, (intracellular hydrogen peroxide (flow cytometry), lipid peroxidation products, and aconitase inactivation/reactivation (the latter a reduction-dependent process)] will be measured in these cells before, [during] and after oxidant challenge. Likewise, cystine uptake, as well as activity of the transsulfuration pathway which produces cysteine for glutathione synthesis will be estimated. In addition, efflux of glutathione and release of hydrogen peroxide into the extracellular space will be quantified. Increased G6PD expression will be demonstrated by Northern analysis and activity assays. Because antioxidant enzymes may be feedback regulated by cellular redox status, which can be modulated by G6PD and NADPH, the effect of increased G6PD expression on activities and mRNA's of other antioxidant enzymes including superoxide dismutases will be assessed. In in vivo work, pulmonary histopathologic damage and inflammation, vascular permeability, oxidation of glutathione and NADPH, lipid peroxidation, and mortality due to hyperoxia will be quantitated in adult transgenic mice with increased expression of G6PD in lung and in appropriate control mice. [In mice exposed to hyperoxia during the neonatal period, the effect of elevated G6PD activity on the inhibition of lung growth, as measured by prevention of DNA accretion, will be studied.] Finally, the effects of increased G6PD expression combined with increased expression of CuZnSOD and/or MnSOD in mice in both adult and newborn lung injury models will be studied. These experiments will provide an improved understanding of important antioxidant defenses which protect against peroxidative injury in the lung.